Synergistic interactions of assorted ameliorating agents to enhance the potential of heavy metal phytoremediation

Pollution by toxic heavy metals creates a significant impact on the biotic community of the ecosystem. Nowadays, a solution to this problem is an eco-friendly approach like phytoremediation, in which plants are used to ameliorate heavy metals. In addition, various amendments are used to enhance the potential of heavy metal phytoremediation. Symbiotic microorganisms such as phosphate-solubilizing bacteria (PSB), endophytes, mycorrhiza and plant growth-promoting rhizobacteria (PGPR) play a significant role in the improvement of heavy metal phytoremediation potential along with promoting the growth of plants that are grown in contaminated environments. Various chemical chelators (Indole 3-acetic acid, ethylene diamine tetra acetic acid, ethylene glycol tetra acetic acid, ethylenediamine-N, N-disuccinic acid and nitrilotri-acetic acid) and their combined action with other agents also contribute to heavy metal phytoremediation enhancement. With modern techniques, transgenic plants and microorganisms are developed to open up an alternative strategy for phytoremediation. Genomics, proteomics, transcriptomics and metabolomics are widely used novel approaches to develop competent phytoremediators. This review accounts for the synergistic interactions of the ameliorating agent’s role in enhancing heavy metal phytoremediation, intending to highlight the importance of these various approaches in reducing heavy metal pollution.


Introduction
The rate of heavy metal pollution is increasing daily due to the uncontrolled discharge of sewage sludge and mining waste, excess use of chemical fertilizers and pesticides, intuitive industrial activities, etc. (Ekta and Modi 2018).At the same time, the macro and micro nutrients essential for plant growth include some heavy metals like cobalt (Co), copper (Cu), chromium (Cr), iron (Fe), nickel (Ni), manganese (Mn) and zinc (Zn).However, a higher concentration of these heavy metals imparts stress to the plants.There are some non-essential heavy metals like cadmium (Cd), mercury (Hg), arsenic (As), lead (Pb), etc. which are highly lethal to living organisms (Bhat et al. 2022).The bioavailability and toxicity of heavy metals mainly depend on the forms in which they are present in the environment (Shi et al. 2022).The physiochemical processes such as ion exchange, precipitation, reverse osmosis, evaporation and chemical reduction are the remediation methods of heavy metal-polluted soil.However, the main problem is that these methods require external man-made resources and are too costly.
Plants play an important role in counteracting the harmful effects of these assorted pollutants to a greater extent in many ways.One promising aspect is the phytoremediation, which acts as a green surrogate to minimize the level of toxic metal ions with the employment of plants.Phytoremediation is an eco-friendly and low-cost method that can address the problems of metal pollution in a sustainable manner (Ekta and Modi 2018).In this emerging phytotechnology, various metal-tolerating plants are exploited for their ability to clean polluted soil or water.
In addition, some microorganisms, algae and other lower groups of plants, genetically engineered plants, etc., also help enhance the phytoremediation efficiency.The plant-microbe interaction is vital to proper plant growth, development and soil health (Rawool et al. 2020).In addition to this, some microorganisms like phosphate-solubilizing bacteria, endophytic microorganisms, mycorrhizas and plant growth-promoting rhizobacteria also exhibit the ability of heavy metal tolerance (Sruthi et al. 2016;Rawat et al. 2021;Sharma et al. 2021a, b).Nowadays, chemicals like Ethylene Diamine Tetra Acetic acid (EDTA), Ethylene Glycol Tetra Acetic acid (EGTA), Indole 3-Acetic Acid (IAA), Ethylenediamine-N, N′-disuccinic acid (EDDS) and Nitrilotri-acetic acid (NTA) are commonly used in order to enhance the efficiency of heavy metal phytoremediation.These chemicals interact with the rhizosphere and increase the mobility and metal uptake of plant roots.The synergistic interactions and cross-talk of soil microorganisms and various chemical chelators with the plant roots, which play a vital role in alleviating the stress and promoting the phytoremediation process, are detailed in the following sections.

Phytoremediation-an overview
Phytoremediation refers to a diverse assemblage of work-based technologies that use naturally occurring or genetically engineered plants to clean contaminated environments (Flathman and Lanza 1998).The word 'Phytoremediation' is derived from the Greek prefix "phyto", which means plant, and the Latin suffix "remedium", which means clean or restore (Cunningham et al. 1997).Different plant-based technologies are grouped under phytoremediation-phytoextraction, phytostabilization, rhizofiltration, phytodegradation and phytovolatilization.
Phytoextraction is a method in which the metal absorbed by the plant is translocated to harvestable shoots and accumulates there.Phytostabilization differs from the rhizofiltration method in that it uses the plant to stabilize the contaminated soil rather than cleaning it.In contrast, in rhizofiltration, the plants are used to clean various aquatic environments.On the other hand, phytodegradation uses plants to uptake, metabolize and degrade the organic contaminants.Some plants extract certain volatile metals from soil and release them into the atmosphere through volatilization, known as phytovolatilization (Vasavi et al. 2010;Ekta and Modi 2018).
Plants chosen for phytoremediation should be fastgrowing, high biomass yielding, high metal tolerating and hyperaccumulating, easily cultivatable and harvestable (Vasavi et al. 2010).There are many plants around us which have good phytoremediation potential and play a significant role in reducing the harmful effects of heavy metal pollution by performing various detoxification mechanisms like exclusion, excretion, accumulation, enzymatic and non-enzymatic anti-oxidant mechanisms, accumulation of osmoprotectants, chelation of metal by metallothioneins and phytochelatins, etc. (Sruthi et al. 2016).

Symbiotic microorganism
Microorganism-based bioremediation is a safe, inexpensive and eco-friendly option, and soil microbes promote/ oppose or inhibit diverse biotic and abiotic processes, thereby sustaining the soil ecosystem (Dongre 2021).Plant microbiome interactions efficiently enhance the demolition of contaminants, incredibly toxic heavy metals, from the ecosystem by improving trace element uptake and translocation (Mocek-Plociniak et al. 2023).The microorganisms also assist the growth of plants grown in contaminated environments and mainly comprise phosphate-solubilizing bacteria (PSB), endophytic microorganisms, mycorrhizal associations and plant growth-promoting rhizobacteria (PGPR).

Phosphate-solubilizing bacteria (PSB)
During stressed conditions, using phosphate-solubilizing microorganisms is an eco-friendly approach to maintain agro-environmental sustainability.These microorganisms solubilize both inorganic and organic phosphorus through various mechanisms such as the production of organic acids, inorganic acids, hydrogen sulfide, siderophores, protons, excretion of extracellular enzymes; direct oxidation pathway and also through enzymatic actions (Yadav 2022).Additionally, these microbes not only solubilize phosphate but also promote plant growth and crop yield by producing plant growthpromoting hormones like auxins, gibberellins and cytokinins, antibiosis against pathogens, ACC deaminase (1-aminocyclopropane-1-carboxylate deaminase) which enhances plant growth under stress conditions, etc. thereby improving plant resistance to heavy metal toxicity (Rawat et al. 2021).
PSB abundance enhances the Cu-amelioration capacity of Wedelia trilobata, performing a higher rate of Cu absorption and translocation from contaminated soil (Lin et al. 2018).In high-Cd-mobilizing PSB, gluconic acid produced due to the peripheral peroxidation pathway is mainly responsible for high-Cd-dissolution.In low-Cdmobilizing PSB, glycolic acid plays that role (Yang et al. 2018).For the remediation of Pb-contaminated sediments, PSB capsules coupled with phosphate materials are the most effective method (Zhang et al. 2020).
Along with a significant role in promoting various plant growth parameters, phosphate-solubilizing microorganisms also help to solve excess phosphorus contamination in phosphate mining wastelands by improving phytoremediation efficiency (Guo et al. 2021).The combined action of PSB and biochar-supported nano-hydroxyapatite results in the immobilization of Cd in contaminated river sediments and is a promising candidate for passivation material for sediment Cd (Zhao et al. 2022).Similarly, the application of PSB together with rice husk biochar in the soils that were heavily polluted by heavy metals like Pb/Cd helps in the reduction of soil acidification, enhancement of nutrients in the soil as well as total biomass of microbes within a short duration, that may also account for the reduced diffusion of toxic heavy metals (Lai et al. 2022).The application of PSB and plant growth-promoting rhizobacteria (PGPR) in Vigna radiata helps to reduce Cr toxicity to a great extent and also causes a tremendous increase in leaf number and area, length of root and shoot and chlorophyll synthesis (Mohanty and Mohapatra 2023).

Endophytic microorganism
The group of microorganisms colonized in the interior part of the plant, such as the root, stem or seeds and do not have any adverse effect on the host plant are referred to as endophytes (Rawool et al. 2020).Nowadays, endophytic microbes are explored mainly for their role in heavy metal stress mitigation in plant systems (Sharma et al. 2021a).Endophytic fungi provide enormous services to their host plants, including growth enhancement by nutrient acquisition, detoxification of heavy metals, secondary metabolite regulation and enhancement of abiotic/biotic stress tolerance (Khalid et al. 2021).
Endophytes isolated (Enterobacter species-LC1, LC4 & LC6; Kocuria species-LC2, LC3 & LC5 and Kosakonia species-LC7) from Lantana camara established within Solanum nigrum effectively improved plant growth besides increasing bioaccumulation and root to shoot transport of As when applied as consortium (Mukherjee et al. 2018).Endophytic bacterium Sphingomonas SaMR12 isolated from Cd/Zn hyperaccumulator Sedum alfredo improves the plant phytoextraction efficiency and heavy metal (Cd) tolerance.Hence these endophytes are considered an effective remediation candidate (Wang et al. 2020c).Some endophytic bacterial inoculation leads to the expression of heavy metal ATPase genes (HMAs) that encode heavy metal transport proteins.In rice seedlings, HMA2, HMA3, and HMA4 play a leading role in Cd translocation (Ullah et al. 2022) (Table 1).

Mycorrhizal association
Some fungi, namely mycorrhizas, are symbiotically associated with the roots of higher plants, and this positive interaction enhances water and nutrient uptake from the soil, thereby enhancing growth and yield.Mycorrhizal fungi form their extensive hyphal network in soil, and the extra-radical mycelia (ERM) serves as an artificial root system to increase nutrient uptake (Mahadevakumar and Sridhar 2020).Mycorrhizae comprises seven types of members: arbuscular, ecto, ectendo, arbutoid, monotropoid, ericoid and orchidaceous.Arbuscular and ectomycorrhizae are the most abundant and ubiquitous types (Parihar et al. 2020).Arbuscular mycorrhizal fungi (AMF) are associated with heavy metal absorption and tolerance in plants and hence act as stress alleviators by bioremediating soil polluted with heavy metal (Sruthi et al. 2016).The distinct feature of AMF is arbuscules, the nutrient exchange site between the host and fungi, which are also involved in metal uptake (Parihar et al. 2020).
The application of AMF, along with soil amendments, becomes the most effective strategy for heavy metal phytoremediation (Wang et al. 2022a).Similarly, inoculation of mycorrhiza and fly ash in Acacia luederitzii influences the dry matter accumulation by reducing the heavy metal (Cu, Ni, Pb, Mn and Zn) availability and metal uptake (Ultra and Manyiwa 2021).Various biochemical processes like metal detoxification, metal mobilization or immobilization, accumulation, transformation and translocation are facilitated by AMF, showing their beneficiary role in phytoremediation (Tiwari et al. 2020).The earthworm-AMF-plant symbiosis potentially plays an essential role in the phytoremediation of heavy metal-polluted soils (Wang et al. 2020a).AMF inoculation plays a vital role in the environment contaminated with As, Cd, Pb and Cr, in which AMF increases the accumulation rate of these metals in the roots of plants and increases the resistance of plants to the high toxicity of these metals, showing its enhanced phytoremediation efficiency (Boorboori and Zhang 2022).Moreover, the application of AMF and biochar in maize grown in the soils artificially contaminated with 5 mg Cd Kg −1 soil, is suitable for phytoremediation of Cd without much deleterious effects to the plant (Zhuo et al. 2020).At the same time it was also proved earlier that, in situations where the soil Cd concentration is 25 mg Kg −1 , the ornamental plant Mirabilis jalapa can be successfully employed for the remediation of Cd contaminated soil (Wang and Liu 2014) (Table 2).

Plant growth promoting rhizobacteria (PGPR)
PGPR is a group of rhizobacteria that enhance plant growth and improve yield by producing various plant growth-promoting substances.PGPR is a biofertilizer and bioprotectant (Mahadevakumar and Sridhar 2020).PGPR has two modes of action-direct and indirect.Nitrogen fixation, phosphate solubilization and phytohormone production belong to the direct action.The indirect mechanism protects plants from plant pathogens by producing antimicrobial compounds (Glick 1995;Martinez-Martinez et al. 2023).Phosphate-solubilizing PGPR amends phytoextraction and phytostabilization efficiency of heavy metal treated plants.Using ACC deaminase activity, PGPR also enhances the growth of plants even in the presence of heavy metals (Kumar et al. 2023;Martinez-Martinez et al. 2023).
Multifunctional Plant growth promoting bacteria (PGPB)/PGPR showed Cr resistance and bio-inoculant properties with phytoremediation plants.By modifying root architecture and sequestering metals in the rhizosphere, PGPB enhances Cr uptake and lessens phytotoxicity.Growth regulators, mineral solubilizers, phytohormones and diverse secondary metabolites were produced by PGPB in order to speed up plant defence against metal poisoning (Dongre 2021).The phytoremediation efficiency of ryegrass on Cu-Cd co-contaminated soil can be improved by applying PGPR (Shi et al. 2022).The combined action of PGPR and salicylic acid (SA) in sunflowers helps to improve the heavy metal (Cd, Pb and Ni) phytoremediation efficiency and plant growth (Khan et al. 2018).PGPR adopt various defence mechanisms against heavy metal stress, such as compartmentalization, exclusion, complexity rendering, and the synthesis of metal-binding proteins (Sharma et al. 2021b) (Table 3).

Chemical chelators
The application of various chemicals is a promising approach to heavy metal phytoremediation when the heavy metal extraction by the plant is limited or poor (Hasan et al. 2019).When using chemical chelators for heavy metal remediation, the most crucial things considered are the plant type, application rate and chelate types (Baghaie and Polous 2019).Competition with other cations must also take into consideration when applying chemical chelators (Wang et al. 2022c).Several metalchelating chemical agents have been supplemented in the soil to enhance the rate of metal detoxification (Table 4).

Indole 3-Acetic Acid (IAA)
IAA is a plant growth regulator and is the natural form of auxin.It plays a significant role in improving plant growth and heavy metal phytoremediation potential.IAA induces the activation of ATPases in the plasma membrane, thereby producing changes in the transport of ions through the membrane, which are related to heavy metal accumulation (Ji et al. 2015).
For instance, exogenous application of IAA results in the rise of Cd immobilization in Typha latifolia root.The effect may be due to IAA-induced increased synthesis of cell wall components upon which Cd fixation occurs (Rolon-Cardenas et al. 2022).Upon Cd stress, exogenous IAA application enhanced peroxidase

Ethylene Diamine Tetra Acetic acid (EDTA)
EDTA is the most commonly used and potential organic ligand that immobilizes heavy metals, enhances the uptake of metals through roots in the form of soluble metal-EDTA complexes, and supports metal xylem loading (Hasan et al. 2019).EDTA shows a complex relationship with pH, and its metal detoxification efficiency is related to soil types (Subasic et al. 2022).
The application of EDTA, along with hormones like IAA and kinetin, reduced the adverse effects of Cd by increasing the total protein content and peroxidase activity (Shirkhani et al. 2018).EDTA enhances Pb's availability, absorption and translocation in bamboo plants growing in Pb-contaminated soil (Jiang et al. 2019).The most advantageous approach for the remediation of Pb-contaminated soil is using EDTA and biochar because their combined action enhances the phytoextraction rate of Pb and promotes plant growth (Rathika et al. 2021).The environmental risk associated with excess EDTA application can be lowered by the co-action of EDTA with degradable chelating agents like nitrilotri-acetic acid (NTA), and their combined action also enhanced Pb remediation efficiency in the dwarf bamboo plants (Yang et al. 2022b).

Ethylene Glycol Tetra Acetic Acid (EGTA)
EGTA plays a positive role in plant heavy metal uptake and is widely used as a chelating agent (Hasan et al. 2019).EGTA plays a crucial role in Cd accumulation and can be enhanced by applying EDTA (Dai et al. 2020).EGTA show better performance than EDTA in Cd phytoextraction of the ornamental plant Mirabilis jalapa (Wang and Liu 2014;Wei et al. 2018).The application of exogenous EGTA and Ca in chickpea seeds alleviated Cd-induced growth damage and decreased lipid peroxidation and protein carbonylation in both shoots and roots (Sakouhi et al. 2016).The mechanisms induced by Ca and EGTA to protect the cell from Cd-induced oxidative injury include the triggered thiol-protecting process through activation of the Trx system and restoring the control level of antioxidative enzyme activities (Sakouhi et al. 2018).EGTA enhanced Cd accumulations in the dead leaves of tall fescue plants, which could be associated with the increase of the water-soluble inorganic Cd and Cd organic acid complexes in the shoots (Wang et al. 2019).
Upon supplementation of biodegradable chelates such as EGTA, EDDS, NTA and citric acid (CA) in Solanum nigrum, the EGTA application shows improved Cd phytoextraction efficiency compared to others, with an increased tolerance index value, transfer coefficient of root and translocation factor (Sharma et al. 2022b).For the removal of Cr(III) in a highly saline organic wastewater environment, EGTA-modified magnetic microspheres were used (Wang et al. 2020b).

Ethylenediamine-N, N′-succinic acid (EDDS)
EDDS is a biodegradable solid chelating agent.EDDS is produced by biological methods such as fermentation (in Amycolatopsis japonicum MG417-CF17) through the most economical, eco-friendly enzymatic methods in which ethylene diamine and fumaric acid is used as substrate (Wang et al. 2022d).When comparing the efficiencies of EDDS and NTA in enhancing the Zn phytoremediation by alfalfa, EDDS seems more efficient due to the higher Zn concentration in soil pore water induced by EDDS.EDDS can remediate uranium (U) and Cd in Zebrina pendula.However, it is equally efficient in Cd phytoextraction because of its more significant effect on shoot Cd accumulation.The ability of EDDS to activate Cd in soil was better than that of citric acid and oxalic acid treatments (Chen et al. 2019a).
The best amendment combinations for Pb phytoextraction are EDDS and vermicompost (Moslehi et al. 2019).When applied with 5-aminolevulinic acid (ALA), EDDS promoted Cd absorption and biomass accumulation in sunflowers growing on Cd-contaminated soil (Xu et al. 2021).The highest EDDS application leads to lower biomass production in alfalfa.Hence in order to minimize phytotoxicity and improve Zn phytoextraction efficiency in alfalfa, the EDDS dosage should be adjusted for each soil, depending on its characteristics and metal content (Wang et al. 2021).The application of plant growth regulators (diethyl aminopurine and 6-benzylaminopurine) along with EDDS mitigates the negative impact of EDDS   on plant growth, resulting in enhanced Cd phytoaccumulation and translocation (Li et al. 2018).

Nitrilotriacetic acid (NTA)
NTA is an environmentally friendly, biodegradable chelating agent that strengthens phytoremediation (Pu et al. 2022).NTA is a derivative of EDTA.The biodegradable nature and reduced toxicity of NTA towards microorganisms and plants make it more advantageous in phytoextraction techniques (Hart et al. 2022).The lag phase for the degradation of NTA varied from 0-7 days (Wang et al. 2022c).NTA play a vital role in mineral absorption and transportation in centipede grass, showing increased root Mg, K and Ca and shoot Fe, Cu and Mg concentrations (Pu et al. 2022).The chelating capability of NTA makes NTA-modified Dendrocalamus strictus charcoal powder a sound absorbent for the removal of Cu(II) ions from an aqueous solution (Saini et al. 2020).When NTA is applied along with EDTA, Pb remediation efficiency in dwarf bamboo gets boosted significantly (Yang et al. 2022b).The combined action of NTA and Triton-X-100, an alkyl polyglucoside (APG), increased the Pb concentration to more than double that in the foliage of switchgrass (Hart et al. 2022).The NTA application in Athyrium wardii modifies plant rhizosphere by lowering pH, increasing dissolved organic carbon, exudation and soil enzyme activities, These alterations contributed to the increased Pb accumulation (Zhang et al. 2021).

Synergistic impact of chemicals and microbes in heavy metal stress tolerance
The independent application of chemical chelators and microbes for heavy metal tolerance is beneficial, but further improvement is possible through the synergic treatment of both chemical chelators and microbes.The synergistic application of chemical chelators and microbes aided in improving the bioavailability of some toxic metals as well as the microbial population of soil.The simultaneous application of AM fungus and EDTA improved the heavy metal tolerance of corn (Zea mays) and sunflower (Helianthus annuus), and Pb extraction was maximum in EDTA-applied soil (Usman and Mohamed 2009).So, synthetic chelators should increase the bioavailability of selective heavy metals, and the following application of microbes enhances the remediation potential.Cronobacter sakazakii-EDTA complex increased the phytoremediation potential of Zea mays L. to remediate Pb-contaminated soils (Menhas et al. 2021).C. sakazaii-EDTA (5 mM EDTA kg −1 ) complex aided the plant in tolerating metal toxicity by improving biomass production, synthesizing photosynthetic pigments, maintaining the water status, and accumulating proline.Enhances the Cu absorption potential of sunflower and increases its tolerance to copper when applied along with EDTA

Shah et al. 2023
Silybum marianum (L.) Gaertn Reduced the Pb uptake Bhardwaj et al. 2023 Moreover, maize plants showed differential tolerance levels towards different soil types, and spiked and aged soil showed different responses under the application of chelators (Menhas et al. 2021).This improvement in the tolerance level depends on the changes in the microbial population due to the soil washing with chelators.Members of Nocardioidaceae were prominent in the soil washed with 10 mmol kg −1 EDTA.However, the dominant microbial population was shifted to chemolithoautotrophic bacteria, such as Nitrososphaeraceae, in the soil washed with 60 mmol kg −1 EDTA (Wei et al. 2020).Thus, knowledge of the nature of the contaminated metal and the microbial population is essential for the better performance of chelators in phytoremediation.

Transgenic approaches
The use of advanced technologies put forward various transgenic approaches to improve the efficiency of heavy metal phytoremediation.The crucial aspects taken into consideration while constructing genetically engineered organisms (GEOs) for the removal of pollutants include modification of enzymes, regulation/control of biological pathways, developments in affinity sensors, post-release monitoring of GEOs, application of molecular tools, risk assessments, pathogenesis, adverse environmental and health effects and biosafety issues (Iravani and Varma 2022).However, in the recent past, several genetically modified organisms have been engineered which have increased potential for metal detoxification without compromising the growth process (Table 5).

Transgenic plants
Plants can be engineered to improve their ability to remediate metal pollution through the transfer and insertion of desirable genes from a foreign source into a plant of interest and produce transgenic plants with overexpression of the desirable genes like genes involved in metal uptake, translocation, sequestration, etc. (Placido and Lee 2022).The advantages of genetic engineering are the requirement of a short period and the ability to transfer desirable genes from hyperaccumulators to sexually incompatible plant species, and these are impossible in traditional breeding methods.While designing transgenic plants, selecting desirable genes and host plants are the main factors considered (Yan et al. 2020).
The merA and merB expressing transgenic plants (Arabidopsis, tobacco, tomato and rice) grown in Hgcontaminated soil can produce safe food like vegetables, fruits and grains for human and animal consumption (Li et al. 2020c).Regulation of the thioldependent mechanism helps to reduce the heavy metal toxicity in Arabidopsis thaliana and is achieved through the overexpression of the MT1 gene (Dubey et al. 2021).SlJMJ524 gene overexpression in Arabidopsis plants controls metal transporter-related gene expression as well as increased flavonoid content in plants, thereby exhibiting Cd tolerance during seedling and maturation stages (Li et al. 2022a).
Similarly, the glutathione derived phytochelatins (PC) molecules are usually synthesized when plants encounter heavy metal stress and its synthesis is mediated by the enzyme phytochelatin synthase (PCS).Their mode of action is in such a way that it binds to free metal ions and sequester it to the vacuoles (Sruthi et al. 2016;Yan et al. 2020;Zhu et al. 2021;Jin et al. 2022).The overexpression of PCS gene has a greater contribution to Cd tolerance in plants by regulating PC synthesis (Zhu et al. 2021;Jin et al. 2022).For example, the overexpression of Boehmerianivea derived PCS gene, BnPCS1 in Arabidopsis showed improved tolerance, accumulation and translocation of Cd along with the reduced cellular damages in these transgenic lines (Zhu et al. 2021).Similarly, the overexpression of maize ZmPCS1 gene in Arabidopsis enhanced Cd tolerance where as its ectopic expression in Arabidopsis mutant lines (atpcs1) helps to overcome the Cd hypersensitivity of atpcs1.Also its transient expression in tobacco reduced Cd toxicity (Jin et al. 2022).
Most of the transgenic plants show high proline content, antioxidant enzyme activities with lower hydrogen peroxide, MDA and decreased electrolyte leakage during heavy metal stress (Kumar et al. 2020;Djemal and Khoudi 2022;Kumar 2022 ;Shahbazi et al. 2022).In addition to the Cd tolerance in transgenic wheat, TaS-WEET14 overexpression alters ion transporter gene expression, and TaSWEET14 expression is positively regulated by TaMYB41 at its transcriptional level.Likewise, AetSRG1 overexpression prevents degradation of phenylalanine ammonia-lyase (PAL) and programmed cell death in Aegilops tauschii (Liu et al. 2022a, b;Wei et al. 2022).

Transgenic microorganism
Genetically engineered microbes (GEM) are constructed using recombinant DNA technology.A desirable gene from an organism of the same or different species is inserted into a microbial genome or plasmid (Verma et al. 2020).The main advantages of genetically engineered microorganisms for their use in heavy metal bioremediation are the cost-effectiveness, ecofriendliness, simplicity and upscalability.The genetically engineered bacteria can improve metal-chelating proteins, metal stress tolerance, bioaccumulation of heavy metal and overexpression of peptides thereby executing bioreduction and recovery of heavy metal ions (Iravani and Varma 2022).Genetic modification of channel proteins (belongs to heavy metal uptake and transport system) and metal binding entities (belongs to heavy metal storage system) like metallothionein (MT), phytochelatins (PC) and polyphosphates (PolyP) enhances heavy metal phytoremediation efficiency (Verma et al. 2020).
Genetically engineered bacteria expressing MT have been increasingly used to treat heavy metals.MT is a low molecular weight cysteine-rich proteins that enable them to readily bind and sequester metal ions (Tsyganov et al. 2020;Li et al. 2021;Uckun et al. 2021).The use of transgenic rhizobia in association with legumes to enhance phytoremediation efficiency is collectively called symbiotic engineering (Jach et al. 2022).The Cd removal rate of transgenic E. coli (MT3 and MT2) is affected by temperature, pH and contact time (Uckun et al. 2021).In the genetically engineered E. coli, the ShMT gene is modified by site-directed mutagenesis and recombinant proteins (ShMT1, ShMT2 and ShMT3 having one, two and three-point mutation respectively) were further enhanced using SUMO fusion expression system to yield SUMO-ShMT1, SUMO-ShMT2 and SUMO-ShMT3 having enhanced heavy metal binding capacities (Li et al. 2021).The coassembly of genetically engineered E. coli (SynEc2) and magnetic nanoparticles modified by polyethyleneimine and diethylene triamine pentaacetic acid  captures heavy metals with high removal efficiency (Zhu et al. 2020).Heavy metal biosorption is facilitated by functional groups on the cell membrane of recombinant cells (Jia et al. 2022).

Omics tools
Omics tools are novel approaches to develop competent phytoremediators and the technique may include genomics, proteomics, transcriptomics and metabolomics.Genomics DNA sequencing and analysis are carried out, whereas in proteomics, target protein identification, quantification, and expression analysis takeranscriptomics involves RNA sequencing, expression and regulation profiling.Metabolomics is an implicit tool for profiling metabolites, hormones and signalling molecules (Agarwal and Rani 2022;Anjitha et al. 2023).
Proteomics studies help to understand protein modification during Cd stress (Li et al. 2022b); Cd regulated transport proteins like ABC transporters, ion transport proteins, aquaporins, proton pumps and organic transport proteins (Zhu et al. 2022); protein abundance during Pb exposure such as increased abundance of hemicellulose and pectin related cell wall proteins for sequestration Pb thereby reducing its toxicity (Shen et al. 2021); effect  of exogenous nitrogen on protein expression patterns under Cd stress (Zhang et al. 2022), etc. Gene structure, evolution and phylogenetics, chromosomal localization, gene doubling, cis-elements and expression profiles of genes during heavy metal stress were determined using genomics and bioinformatics (Gao et al. 2022;He et al. 2022a;Xie et al. 2022).
Transcriptome analysis reveals that multiple heavy metals co-regulating unigenes exhibited the function of anti-oxidant enzymes, anti-oxidant substances, transporters, transcription factors and cell wall components (Ge et al. 2022).A comparison of the role of potential genes involved in heavy metal detoxification in Calotropis gigantea leaves and root with the aid of comparative transcriptome analysis shows that most of the genes downregulated in the root but up-regulated in the shoot (Yang et al. 2022a).In heavy metal phytoremediation, metabolite accumulation as part of defensive metabolic pathways performs a significant role.Using metabolomics, metabolic profiling helps to understand the alterations in metabolites and metabolic pathways during heavy metal exposure and their role in heavy metal tolerance (He et al. 2022b;Zou et al. 2022;Anjitha et al. 2023) (Table 6; Fig. 1).

Future prospectus
As in view of future prospectus, the upcoming research studies should focus on the advancement to the existing knowledge and data base regarding the tolerant genotypes of plants.Moreover, focus must be also given to tackling the risk regarding the use of certain amendments employed for the enhancement of phytoremediation techniques.Research should also concentrate on how to increase the success rate of transgenic approaches to impart the tolerance towards pollutants.Furthermore, it has been reported that the application of soil amendments helps to improve the soil microflora but it never addressed the query that whether this enhancement in the soil microflora may have any influence on the neighboring plants growing in the soil.

Conclusion
Phytoremediation is a promising method for decontaminating the polluted environment using plants and the remediation efficiency of plants can be enhanced by the application of symbiotic microorganisms, chemicals and various transgenic approaches.Combined applications of chemicals with other amendments practices increase the efficiency of heavy metal phytoremediation than the independent treatments.But the major limitation is that certain chemicals can negatively affect the plant growth, as it causes phytotoxicity above the optimal level.Even though advancement in modern-sustainable techniques leads to the development of noval approaches for the remediation of heavy metals includes application of the microbial community, invention of transgenic organisms and analysis of omics data.With the aid of modern technologies, we can design potential transgenic organisms to alleviate heavy metal toxicity.Comparison omics tools like genomics, proteomics, metabolomics and transcriptomics with genome editing technique aid to evaluate the functional aspects of genes and proteins involved in heavy metal tolerance.These modern technologies can offer plant and microbes with high remediation potential and high metal stress tolerance for the clearing of polluted land in a short duration.

Fig. 1
Fig. 1 Interaction of different biotic as well as abiotic agents for the amelioration of heavy metal stress in plants

Table 1
Role of PSB in alleviating heavy metal phytoremediation potential by plants Zhang et al. 2023tiCd The combined action of fruit peel-based activator & PSB enhanced phytoextraction of Cd and growth of ryegrassZhao et al. 2023bBacillus amyloliquefaciens (CZ-B1) Pb The resistance of CZ-B1 to Pb is mainly achieved by cellular and secretions adsorption under low Pb stressZhang et al. 2023

Table 2
Role of endophytic microorganism in alleviating heavy metal phytoremediation potential by plants

Table 3
Role of mycorrhizae and PGPB in alleviating heavy metal phytoremediation potential by plants

Table 4
Role of various chemical chelators in alleviating heavy metal phytoremediation potential by plants

Table 5
List of transgenic organisms engineered so as to enhance the heavy metal phytoremediation potential

Table 6
Various OMICS tool employed in the field of heavy metal phytoremediation potential in plants